Galenic formulation for colon targeted delivery of active principles

ABSTRACT

Forms of colonic delivery suited to be used orally and designed for colonic delivery of active ingredients selected from the group comprising enzymes capable of inactivating macrolide antibiotics and related compounds, enzymes capable of inactivating quinolones, and β-lactamases.

The present invention relates to a galenic form for colon-targeteddelivery, its preparation process and its use in therapeutic.

Specific release systems in the colon have been proven to havesignificant theracanic advantages.

A large number of colonic illnesses could effectively be treated moreefficaciously if the active ingredient were released locally. This isthe case, inter alia, for Crohn's disease, ulcerative colitis,colorectal cancer and constipation.

Colon-targeted release could also be interesting when, from thetheracanic point of view, a delay in absorption is necessary, inparticular in the treatment of pathologies such as nocturnal asthma orangor (Kinget R. et al. (1998), Colonic Drug Targeting, Journal of DrugTargeting, 6,129).

The administration of polypeptidic active ingredients occurs essentiallyparenterally, which is painful and the origin of poor observation oftreatment. For some years now there has been an interest in using thecolon as an absorption site for peptidic active ingredients (analgesic,contraceptive, vaccine, insulin . . . ). The absorption of peptides inthe colon seems effectively better than at other sites of the digestivetract, in particular due to proteolytic activity clearly weaker than inthe small intestine and in the absence of peptidasic activity associatedwith the membrane of the epithelial colonic cells.

During oral administration of antibiotics, these pass through thestomach and are then absorbed in the small intestine to diffuse in theentire organism and treat the infectious focus for which they have beenadministered. All the same, a fraction of ingested antibiotics (whereofthe importance varies with the characteristics peculiar to each type ofantibiotic) is not absorbed and continues its progress to the colonbefore being eliminated in the stool. These residual antibiotics arereunited, in the small intestine, by a fraction of the antibioticsabsorbed, but which are re-excreted in the digestive tract by way ofbiliary elimination. This fraction is of variable importance as afunction of metabolism and elimination paths of each antibiotic.Finally, for certain antibiotics, a fraction of the dose absorbed iseliminated directly by the intestinal mucous in the lumen of thedigestive tract. Since the antibiotics had been administered orally orparenterally, an active residual fraction is generally found in thecolon. This holds true, to varying degrees, for the vast majority of thefamilies of antibiotics utilised in therapeutics, the only notableexception being the family of amino-glycosides for which intestinalexcretion is negligible. For the other antibiotics, intestinal excretionof a residual antibiotic activity will have different consequences, allharmful. In effect, in the colon there is a complex (several hundreds ofdifferent bacterial species) and very dense (more than 10¹¹ bacteria pergram of colonic content) bacterial ecosystem which will be affected bythe arrival of active residues of antibiotics. The following isobserved:

-   -   1) imbalance of flora which would be the main cause of banal        diarrhoea sometimes following ingestion of antibiotics        (Bartlett J. G. (2002) Clinical practice. Antibiotic associated        diarrhea, New England Journal of Medicine, 346, 334). Even        though this diarrhoea is generally not serious and quickly        abates, either spontaneously, or when treatment is discontinued,        it is nevertheless badly received by patients and adds to the        discomfort of the basic illness for which antibiotics have been        prescribed;    -   2) perturbation of the functions of resistance to colonisation        by exogenic bacteria (or “barrier effect”) with the possibility        of increased risk of infection, for example, alimentary        intoxication to Salmonella (Holmberg S. D. et al. (1984) Drug        resistant Salmonella from animals fed antimicrobials, New        England Journal of Medicine, 311, 617);    -   3) selection of micro-organisms resistant to antibiotics. The        latter can be of various types:        -   a) they can first be pathogenic bacteria, such as for            example Clostridium difficile, a species capable of            secreting toxins causing redoubtable colitis known as            pseudomembranous (Bartlett J. G. (1997) Clostridium            difficile infection: pathophysiology and diagnosis, Seminar            in Gastrointestinal Disease, 8,12);        -   b) they can also be relatively slightly pathogenic            micro-organisms but whereof the multiplication can lead to            surrounding infection (vaginal Candidosis or Escherichia            coli resistant cystitis).        -   c) they can finally be non-pathogenic commensal resistant            bacteria but whereof the multiplication and faecal            elimination will boost dissemination in the environment. As            it is, these resistant commensal bacteria can constitute an            important source of resistance mechanisms for pathogenic            species. This risk is currently considered to be major in            terms of the worrying character in the evolution to            multiresistance of numerous pathogenic species for humans.

Numerous strategies exploiting the diverse physiological parameters ofthe digestive tract have therefore been envisaged with a view toreleasing active ingredients in the colon. Studies have in particularbeen carried out by means of administration systems based on (1)utilisation of polymers sensitive to variations in pH, (2) release formsdependent on time, (3) prodrugs or again polymers degradable by bacteriain the flora.

(1) Systems Based on Variations in pH.

The pH in the stomach is of the order of 1 to 3 but it increases in thesmall intestine and colon to reach values close to 7 (Hovgaard L. et al.(1996) Current Applications of Polysaccharides in Colon Targeting,Critical Reviews in Theracanic Drug Carrier Systems, 13,185). For anactive ingredient to reach the colon, without undergoing thesevariations in pH, it is possible to administer it in the form oftablets, gels or spheroids coated in a pH-dependent polymer, insolublein acid pH but soluble in neutral or alkaline pH (Kinget et al. op cit).The most commonly used polymers are derivatives of methacrylic acid,Eudragite L and S (Ashford M. et al. (1993), An in vivo investigationinto the suitability of pH-dependent polymers for colonic targeting,International Journal of Pharmaceutics, 95,193 and 95,241; and David A.et al. (1997) Acrylic polymers for colon-specific drug delivery, S.T.P.Pharma Sciences, 7, 546).

Given the important inter- and intra-individual variability of thevalues of pH at the gastro-intestinal tract level, pH-dependent polymersdo not represent the best means for obtaining specific release in thecolon (Ashford M. et al., op cit.).

(2) Systems Based on Transit Time.

The formulation of these systems is such that it allows release of theactive ingredients after a predefined lag time. So as to release theactive ingredient in the colon, these forms must still resist the acidenvironment of the stomach and enter a silent phase of a predeterminedtime, before releasing the active ingredient, corresponding to thetransit time from the mouth to the terminal ileon (Gazzaniga A. et al.(1995) Time-dependent oral delivery systems for colon targeting, S.T.P.Pharma Sciences, 5,83 and 108, 77; Liu P. et al. (1999)Alginate/Pectin/Poly-L-lysine particulate as a potential controlledrelease formulation, J. Pharm. Pharmacol., 51, 141; Pozzi F. et al.(1994) The Time Clock system: a new oral dosage form for fast andcomplete release of drug after predetermined lag time, Journal ofControlled Release, 31,99).

Pulsincap® by Scherer was one of the first formulations of this type(international patent application WO90/09168). It has the appearance ofa gel whereof the body is insoluble in water. The active ingredient ismaintained in the body by a hydrogel stopper placed in the head of thehydrosoluble gel. The whole is coated in a gastro-resistant film. Afterdissolution of the head in the small intestine, the stopper swells oncontact with digestive juices. When the latter reaches a criticalswelling threshold it is ejected, thus allowing release of the activeingredient. The ejection time is controlled by the properties of thehydrogel constituting the stopper.

Systems based on transit time nevertheless offer numerous disadvantages(variations in time for emptying of stomach and transit time, retentionphenomena in the ileo-caecal valve (Kinget R., op. cit.), causing a lackin specificity and preventing validation of the latter as specificrelease systems in the colon. Finally, large-scale production of thistype of system is difficult to envisage, as this would require andcostly significant adaptation of industrial technologies.

Recently a novel form for colonic targeting has been developed,“Colon-targeted Delivery Capsule” (CTDC) (Ishibashi T. et al. (1998)Design and evaluation of a new capsule-type dosage form forcolon-targeted delivery of drugs, International Journal ofPharmaceutics, 168,31 and 57,45). The CTDC is a system bringing togetherthe pH-dependent factor and the time-dependent factor. It is in the formof a classic gel encasing the active ingredient and an organic acid(succinic acid), covered in 3 layers.

(3) Systems Based on Enzymatic Activity of the Microbial Colonic Flora.

3.1. Prodrugs.

Prodrugs have largely been studied for colonic targeting of variousactive ingredients (anti-inflammatory non-steroidal and steroidal,spasmolytic, . . . ). These systems are based on the capacity of theenzymes produced by the colonic flora for degrading prodrugs in order torelease the active form of the active ingredient.

Numerous prodrugs based on the action of the bacterial azoreductases inparticular have been developed with the aim of releasing in the colonactive ingredients such as 5-aminosalicylic acid (5-ASA) utilised in thetreatment of local pathologies such as Crohn's disease or ulcerativecolitis (Peppercorn M. A. et al. (1972) The role of intestinal bacteriain the metabolism of salicylazosulfapyridine, The Journal ofPharmacology and Experimental Therapeutics, 181, 555 and 64, 240).

Another approach consists of exploiting bacterial hydrolases such asglycosidases and polysaccharidases (Friend D. R. (1995) Glycosideprodrugs: novel pharmacotherapy for colonic diseases, S.T.P. PharmaSciences, 5, 70 Friend D. R. et al. (1984) A colon-specificdrug-delivery system based on drug glycosides and the glycosidases ofcolonic bacteria, Journal of Medicinal Chemistry, 27, 261; Friend D. R.et al. (1985) Drug glycosides: potential prodrugs for colon-specificdrug delivery, Journal of Medicinal Chemistry, 28, 51; and Friend D. R.et al. (1992) Drug glycosides in oral colon-specific drug delivery,Journal of Controlled Release, 19, 109). Prodrugs have thus beendeveloped by coupling, for example, steroids to sugars (glucose,galactose, cellobiose, dextran (international patent applicationWO90/09168)), cyclodextrins (Hirayama F. et al. (1996) In vitroevaluation of Biphenylyl Acetic Acid-p-Cyclodextrin conjugates ascolon-targeting prodrugs: drug release behavior in rat biological media,Journal of Pharmacy and Pharmacology, 48, 27).

3.2. Coating by Polymers Biodegradable by Bacterial Enzymes.

In this case, colonic targeting is done by coating the pharmaceuticalform with a polymer specifically degraded by the enzymes produced bymicroflora, by benefiting from the presence of azoreductases orbacterial glycosidases.

Numerous polymers including azoaromatic links have been used to coat anactive ingredient. Saffran et al. (Oral insulin in diabetic dogs,Journal of Endocrinology (1991), 131, 267 and A new approach to the oraladministration of insulin and other peptide drugs, Science (1986), 233,1081) have described the release of insulin and vasopressin in the colonof rats and dogs from oral forms coated with copolymers of styrene andhydroxyethylmethacrylate (HEMA) linked by azoaromatic bonds. Thiscoating is degraded in the colon by bacterial azoreductases responsiblefor release of the active substance.

The advantage of azopolymers is that they allow very good colonicselectivity for release of active ingredients. The disadvantageassociated with use is the lack of information on their possibletoxicity.

To avoid this disadvantage, other studies have chosen to focus on theuse of coating film based on natural substance such as polysaccharidesin particular with coating films based on amylose/ethylcellulose(Milojevic S. et al. (1996) Amylose as a coating for drug delivery tothe colon: preparation and in vitro evaluation using 5-aminosalicylicacid pellets, Journal of Controlled Release, 38, 75), based on dextraneester (Bauer K. H. et al. (1995) Novel pharmaceutical excipients forcolon targeting, S.T.P. Pharma Sciences, 5, 54) or pectin.

3.3. Matrices Biodegradable by Bacterial Enzymes.

Another approach of systems of specific release in the colon consists ofthe elaboration of matrices by compression of a mixture of activeingredient and biodegradable polymers such as chondroitin sulfate(Rubinstein A. et al. (1992b) Chondroitin sulfate: a potentialbiodegradable carrier for colon-specific drug delivery, InternationalJournal of Pharmaceutics, 84, 141 and Rubinstein A. et al. (1992a)Colonic drug delivery: enhanced release of Indomethacin fromcross-linked chondroitin matrix in rat cecal content, PharmaceuticalResearch, 9,276), guar gum (Krishnaiah Y. S. R. et al. (1998) Evaluationof guar gum as a compression coat for drug targeting to colon,International Journal of Pharmaceutics, 171,137), chitosan (Tozaki H. etal. (1997) Chitosan capsules for colon-specific drug delivery:improvement of insulin absorption from the rat colon, Journal ofPharmaceutical Sciences, 86,1016) or pectin (Rubinstein A. et al. (1993)In vitro evaluation of calcium pectinate: a potential colon-specificdrug delivery carrier, Pharmaceutical Research, 10, 258).

Systems based on enzymatic activity of microbial flora are probablythose having the greatest colonic specificity for release of the activeingredients. Therefore they make up a future path for colonic targeting.

The interest in polysaccharides in the preparation of systems forcolonic administration is that they are of natural origin, only slightlytoxic and specifically degraded by bacterial enzymes of the colonicflora.

Thus, pectin is a polysaccharide isolated from the cellular walls ofsuperior vegetables, widely used in the agro-alimentary industry (as agelling agent or thickener of jams, ices . . . ) and pharmaceutical. Itis polymolecular and polydisperse. Its composition varies according tothe source, conditions of extraction and environmental factors.

Pectins are principally composed of linear chains of acidsα-1,4-(D)-galacturonic, sometimes interspersed with units of rhamnose.The carboxylic groups of galacturonic acids can be partially esterifiedto give methylated pectins. Two types of pectin are distinguishedaccording to their degree of methylation (DM: number of methoxy groupper 100 units of galacturonic acid):

-   -   highly methylated pectin (HM: high methoxy) whereof the degree        of methylation varies between 50 and 80%. It is only slightly        soluble in water and form gels in an acid medium (pH<3.6) or in        the presence of sugars;    -   slightly methylated pectin (LM: low methoxy), with a degree of        methylafion from 25 to 50%. More soluble in water than HM        pectin, it gives gels in the presence of divalent cations such        as Ca²⁺ ions. In fact, Ca2+ ions form “bridges” between        carboxylated groups free of galacturonic acids. The network thus        formed has been described by Grant et al. Under the name of        egg-box model (Grant G. T. et al. (1973) Biological interactions        between polysaccharides and divalent cations: the egg-box model,        FEBS Letters, 32, 195).

There are also amidated pectins. Certain groups of methyl carboxylate(—COOCH₃) can be transformed into carboxamide groups (—CONH₂) bytreatment of pectin by ammonia. This amidation imparts novel propertiesto the pectins, in particular improved resistance to variations in pH.

The pectin is degraded by enzymes originating from superior vegetablesand diverse micro-organisms (mushrooms, bacteria . . . ) includingbacteria of human colonic flora. The enzymes produced by the microfloraare composed of polysaccharidases, glycosidases and esterases.

A galenic form is coated by pectin either via compression (Ashford M. etal. (1993b), An evaluation of pectin as a carrier for drug targeting tothe colon, Journal of Controlled Release, 26, 213), or by pulverisation.Coating by compression is generally completed with pectin alone, whereascompression by pulverisation requires the use of a filmogenic polymer inaddition to the pectin (Milojevic S. et al. (1996) Amylose as a coatingfor drug delivery to the colon: preparation and in vitro evaluationusing 5-aminosalicylic acid pellets, Journal of Controlled Release, 38,75; Wakerly Z. et al. (1996) Pectin/ethycellulose film coatingformulations for colonic drug delivery, Pharmaceutical Research, 13,1210).

Numerous matricial forms based on pectin have likewise been studied.They are generally constituted either by pure pectin, or by its complexwith Ca²⁺ ions, slightly hydrosoluble, calcium pectinate. A matrix ofcalcium pectinate including indomethacin has in particular beendescribed by Rubinstein et al. (1992a) Colonic drug delivery: enhancedrelease of Indomethacin from cross-linked chondroitin matrix in ratcecal content, Pharmaceutical Research, 9, 276) showing better stabilityof the calcium pectinate than the pectin alone in digestive juices,while remaining sensitive to the action of pectinolytic enzymes.

The amidated pectins, more tolerant to variations in pH have also beenstudied for elaboration of matricial tablets for colonic observation(Wakerly Z. et al. (1997) Studies on amidated pectins as potentialcarriers in colonic drug delivery, Journal of Pharmacy andPharmacologyl. 49, 622).

Aydin et al.( (1996) Preparation and evaluation of pectin beads,International Journal of Pharmaceutics, 137,133) were the first toformulate pectin beads according to the ionic gelification method byBodmeier et al. ((1989) Preparation and evaluation of drug-containingchitosan beads, Drug Development and Industrial Pharmacy, 15, 1475 andSpherical agglomerates of water-insoluble drugs, Journal ofPharmaceutical Sciences, 78, 964), who had disclosed beads of alginateand chitosan. Their objective was to incorporate in the beads twodifferent active ingredients, a cationic (atenolol) and an anionic(piroxicam), so as to characterise possible interactions with pectin.They have thus demonstrated that it was possible to form beads with the2 types of active ingredients and that the operational conditions had amajor influence on the properties of the resulting beads.

Sriamornsak used beads of calcium pectinate to establish a system forspecific release of proteins in the colon, by using bovine serum albumin(BSA) having a molecular weight of 66400 Da as protein model(Sriamornsak P. (1998) Investigation on pectin as a carrier for oraldelivery of proteins using calcium pectinate gel beads, InternationalJournal of Pharmaceutics, 169, 213 and (1999) Effect of calciumconcentration, hardening agent and drying condition on releasecharacteristics of oral proteins from calcium pectinate gel beads,European Journal of Pharmaceutical Sciences, 8, 221). He studied theinfluence of different factors of formulation on the properties of theresulting beads, such as their form, their size, the rate ofencapsulation of the BSA and its release kinetics. Sriamornsak hastherefore demonstrated that the pectinate beads of Ca could be employedfor specific release of proteins in the colon. Obtaining an adequaterelease kinetic profile depends principally on the choice of theformulation and operational conditions for preparation of the beads. Noin vitro/in vivo correlation of the release profiles of the encapsulatedactive ingredients has been established.

To boost the stability of the particles along the digestive tract and toavoid any premature release of the encapsulated active ingredient, it ispossible to reinforce the pectin beads by reticulating them with acationic polymer.

Munjeri et al.( (1997) Hydrogel beads based on amidated pectins forcolon-specific drug delivery: the role of chitosan in modifying drugrelease, Journal of Controlled Release, 46, 273) have reticulated pectinbeads amidated with chitosan. They then showed, by comparing thekinetics of dissolution of reticulated forms and of non-reticulatedforms, that the chitosan allowed the release of the active insolubleingredients to be minimised, but did not significantly modify therelease of the hydrosoluble active ingredients. The loss of activeingredient in conditions emulating those of the stomach and the smallintestine can therefore be limited by formation of a complex between thechitosan and the amidated pectin; the reticulated pectin beads stillremain sensitive to the action of the colonic pectinolytic enzymes.

Another reticulating agent, polylysine, has been tested in the presenceof alginate/pectin beads (Liu P. et al. (1999)Alginate/Pectin/Poly-L-lysine particulate as a potential controlledrelease formulation, J. Pharm. Pharmacol., 51,141). The beadsreticulated by the polylysine seem to release less active ingredient inan acid medium (HCl O,1N) than the non-reticulated beads, except in thepresence of highly hydrosoluble active ingredients. The same type ofeffect is found in an alkaline medium (phosphate buffer, pH 7.5) but itis clearly less marked than in acid medium.

International patent application WO 88/07865 suggests administeringbacteria producing β-lactamases in the colon so as to hydrolyse theresidual antibiotics. The micro-organisms utilised are bacteria withstrict anaerobic metabolism, whereof the production and lyophilisationin sufficient quantity to make a drug are difficult. Furthermore, theyare carriers of genes resistant to the antibiotics encoding forβ-lactamases thus engendering a risk of dissemination of these geneswithin the colonic ecosystem and in the environment.

International patent application WO 93/13795 proposes an oral galenicform containing β-lactamases. It can be composed of saccharose particlesof 1 to 2.5 mm in diameter enclosing the β-lactamases or amidase andoptionally an inhibitor of trypsine, said particles being covered by agastroresistant polymer. These particles could well release the enzymein different segments of the digestive tract so that its activity takesplace as required at the desired site in the intestine.

None of the examples comprises experimental data showing that theproposed galenic formulation is effectively capable of releasing theenzyme in an active form at the desired site in the intestine. Inaddition, no proof of the capacity of galenic preparation foreffectively hydrolysing the antibiotic in vivo, nor even in vitro in amedium reproducing the characteristics of the intestinal medium isgiven.

For all these reasons, it is highly desirable to use a system forreducing the quantity of residual antibiotics which reach the colonafter oral or parenteral antibiotherapy, or capable of delivering anactive ingredient directly to the colon.

Therefore, the object of the present invention is multiparticulargalenic forms to be utilised orally and for colonic delivery of activeingredients.

In the sense of the present invention, active ingredient is understoodto mean a substance or composition which is suitable to be utilised intherapeutics or in diagnostics and can be incorporated in the galenicform according to the invention.

The active ingredient can be an anti-infectious, for exampleantibiotics, anti-inflammatory compounds, anti-histamines,anti-cholinergics, antivirals, antimitotics, peptides, proteins, genes,anti-sense oligonucleotides, diagnostic agents and/or immunosuppressiveagents or bacteria.

Examples of particularly advantageous active ingredients areanti-inflammatory agents, antitumoral agents, anti-senseoligonucleotides and enzymes capable of inactivating antibiotics in thecolon, in particular β-lactamases or enzymes capable of inactivatingmacrolids and related substances, such as erythromycin esterasedescribed by Andremont A. et al.((1985) Plasmid mediated susceptibilityto intestinal microbial antagonisms in Escherichia coli Infect. Immun.49 (3), 751) or capable of inactivating quinolones such as thosedescribed by Chen Y et al.( (1997) Microbicidal models of soilmetabolisms biotransformations of danofloxacin, Journal of IndustrialMicrobiology and Biotechnology 19,378).

The active ingredients can be hydrosoluble or liposoluble.

In an advantageous embodiment of the invention, the multiparticulargalenic forms suitable to be utilised orally and for colonic delivery ofactive ingredients comprise pectin beads in the form of a cationic saltenclosing the active ingredient, said pectin being reticulated by acationic polymer.

In an advantageous embodiment according to the invention the cationicpolymer which allows reticulation of the pectin is selected from thegroup composed of polyethylenimine, polylysine, chitosan and theirderivatives.

More advantageously, the molecular weight of these cationic polymers, isbetween 10,000 and 100,000 Daltons, preferably between 20,000 and 50,000Daltons.

In another advantageous embodiment of the invention, the cationic pectinsalt used is calcium pectinate. In the sense of the present invention,pectin is also understood to mean methylated or non-methylated, amidatedor non-amidated pectin.

The galenic forms according to the invention can be administered in alloral forms, in particular in the form of gels and capsules.

These gels and these capsules can be administered simultaneously orsuccessively with other active ingredients, in particular when the gelsor the capsules contain enzymes capable of inactivating the antibiotics,they can be administered simultaneously or successively with thepreparation of corresponding antibiotics.

The active ingredients administered conjointly with the gels andcapsules containing the galenic forms according to the invention areadministered orally or via any other method.

The galenic forms according to the invention can be prepared by methodsknown to the expert or by novel processes which likewise make up part ofthe invention.

Accordingly, the object of the present invention is also a process forpreparation of multiparticular galenic forms, characterised in that anaqueous solution of pectin containing the active ingredient is addeddropwise to a concentration of 0.5 to 5% (v/v) in a solution of calciumchloride to form the beads of calcium pectinate, then the beads ofcalcium pectinate thus obtained are recovered and introduced to anaqueous solution of the polymer cationic.

In an advantageous embodiment of the process, the pectin solution is 4to 10% (m/v), preferably 4 to 7%, the solution of calcium chloride from2 to 10% (m/v) and the solution of polymer cationic from 0.5 to 2%(m/v), said solution of polymer cationic preferably being a solution ofpolyethylenimine.

In an even more advantageous embodiment of the invention the galenicforms are prepared from a solution of pectin at 6% (m/v), a solution ofcalcium chloride at 6% (m/v) and a solution of polyethylenimine at 1% orat 0.6%.

The beads are maintained in the calcium chloride with slow stirring for10 min to 1 hour, preferably for 20 min. The reticulation stage by thecationic polymer is performed with slow stirring for 15 to 40 min,preferably for 20 min.

After recovery of the pectinate beads, the beads are dried at atemperature of between 20 and 40° C. for 30 min to 10 hours, preferablyat 37° C. for 2 hours.

The diameter of the particles according to the invention is between 800and 1500 μm, preferably between 1000 and 1200 μm.

The encapsulation yields are between 50 and 90% or 3-6 UI/beads ofβ-lactamases, activity expressed in the benzylpenicillin substrate,whether the pectin is amidated or not.

Stability in gastric juice is greater than 10 hours and is likewise verygood in intestinal medium USP XXIV, since it is greater than 7 hours(the duration of stability of non-reticulated pectin beads does notexceed 1 hour) and this irrespective of the type of pectin utilised.

Examples 1 to 7 and FIGS. 1 to 8 which follow illustrate the invention.

FIG. 1 shows the effect of reticulation with different concentrations ofPEI (0.6; 0.7; 0.8; 0.9 and 1%) on the disaggregation time of amidatedpectin beads, placed in three different media: PBS, 0.01 M, pH to 7.4;intestinal medium at pH of 6.8+0.1 UPS XXIV; gastric juice at pH of 1.1USP XXIV.

FIG. 2 illustrates the structure of beads containing β-lactamases at therate of 4.4 UI/bead and reticulated for 20 minutes in PEI at 1% andobserved by scanning electron microscopy.

FIG. 3 illustrates the release of in vitro β-lactamases from reticulatedamidated pectin beads prepared according to Example 1 with PEIconcentrations of 0.6 and 0.7% and containing around 5 UI/bead, placedin intestinal medium USP XXIV then in colonic medium (HEPES buffer pH6+pectinolytic enzymes).

FIG. 4 illustrates the evolution of β-lactamase activity in the stoolsof mice as a function of time, after oral administration of reticulatedpectin beads in PEI prepared according to Example 1 and containing 4.4UI/bead.

FIG. 5 illustrates the structure of beads containing β-lactamases at therate of 4.4 UI/bead 30 minutes after in vivo administration. The beadsare then in the stomach. A and B represent the whole beads and C and Dthe cut beads.

FIG. 6 illustrates the structure of beads containing β-lactamases at therate of 4.4 UI/bead 2 hours after in vivo administration. The beads arethen in the small intestine. A and B represent the whole beads and C andD the cut beads.

FIG. 7 illustrates the structure of beads containing β-lactamases at therate of 4.4 UI/bead 4 hours after in vivo administration. The beads arethen in the colon. A and B represent the whole beads and C and D the cutbeads.

FIG. 8 illustrates encapsulation, in pectin beads, of free or complexplasmid DNA with cationic lipids (Lipoplexe) or cationic polymer(Polyplexe).

EXAMPLE 1 Preparation of Galenic Forms

An aqueous solution of pectin at 6% (OF 400 or OG175C Unipectint byDegussa) was introduced dropwise to a solution of calcium chloride at 6%(m/v). The solution of pectin was introduced to the solution of calciumchloride via Tygon piping connected to a peristaltic pump (MicroperpexeLKB Bromma). The solution was passed through a needle of 0.8 mm indiameter (21G, Nedus Terumo) to form drops of pectin which gelledinstantly on contact with the calcium chloride (40 ml) and yielded beadsof calcium pectinate. The beads were kept in the calcium chloride, withslow stirring, for 20 minutes.

The white beads not containing active ingredient (β-lactamases) wereobtained starting out from a solution of amidated (OG 175C) ornon-amidated (OF 400) pectin at 6%. For preparation of loaded beads theactive ingredient (β-lactamases, penicillinases of type A extracted fromBacillus cereus by Sigma) was mixed in with the solution of pectin in aratio of 3% (Vpa/Vpectin).

The resulting beads of calcium pectinate were then recovered byfiltration, rinsed in distilled water, placed on a Petri dish and driedby kiln at 37° C. for 2 hours.

For reticulation in polyethylenimine the undried beads, recovered fromthe solution of CaCl₂ by filtration, were introduced to an aqueoussolution of polyethylenimine (PEI) at 1% and were kept there for 20 minwith gentle stirring.

The beads prepared from the non-amidated pectin OF 400 contained from 1to 2.5 UI/beads and the beads prepared from amidated pectin OG175Ccontained from 1 to 5 UI/beads.

EXAMPLE 2 Stability of Beads

1. Operational Method.

The beads were prepared according to Example 1 with or without thereticulation stage; the duration of reticulation in PEI was 20 minutesin solutions of concentrations ranging from 0.6 to 1%.

The beads were placed either in phosphate buffer (PBS O,01M, pH 7.4), orin media simulating digestive juices (gastric and intestinal USP XXIV)and the disaggregation time was observed.

2. Results.

These are given in FIG. 1.

The beads reticulated or not were stable in the PBS and in the gastricmedium. However, the non-reticulated beads were unstable in theintestinal medium, whereas the beads according to the invention werestable for over 7 hours.

EXAMPLE 3 Morphological Characteristics of Beads

They are illustrated in FIGS. 2A to 2D.

The cuts show that the centre of the beads was full and dense. Thesurface shell corresponds to the PEI. The interior and exterior havedifferent structures.

EXAMPLE 4 Release Kinetics in vitro

1. Operational Method.

Beads reticulated with two different concentrations of PEI (0.6 and0.7%) were prepared according to Example 1 from amidated pectin andcontaining 5 UI/bead. They were left for 5 hours in intestinal mediumUSP XXIV at pH 6.8, then introduced to synthetic colonic medium at pH 6including pectinolytic enzymes (Pectinex Ultra SPL).

The residual β-lactamase activity in the beads was measured over time byspectrophotometry in the presence of nitrocephine.

2. Results.

They are illustrated in FIG. 3.

After 5 hours of incubation beads in intestinal medium (T_(5H)) less 25%of β-lactamase activity which they contain was released. The releasebecomes important in colonic medium under the action of pectinolyticenzymes (Tien), for the reticulated beads with 0.6% PEI, while thesample without pectinolytic enzymes (T_(10H) control) had no significantmodifications of β-lactamase activity. On the contrary, the beadsreticulated with 0.7% PEI did not have their activity diminish after 5Hin colonic medium.

Thus the concentration of PEI modifies the resistance of beads and playson the release time of the active ingredients in colonic medium.

EXAMPLE 5 Release Kinetics in vivo

1. Operational Method.

This assay was performed on male mice CD1. The beads contain 4 UI/bead.

Gels containing 10 beads were administered per os to the mice. Thestools were recovered at time periods of 0, 2 H, 3 H, 4 H, 5 H, 6 H, 7 Hand 8 H and the dosage of β-lactamases in these stools was realized(assay conducted on 5 animals for each time). In addition, one mouse wassacrificed at times of 30 min, 2 H and 4 H so as to recover the beads inits digestive tract and observe their morphological modifications byscanning electron microscopy.

2. Results.

These are illustrated in FIGS. 4 to 7.

The beads arrived intact in the colon after around 3 hours' transit.

The rate of β-lactamases released directly in the stools of micegathered at different times after absorption of the beads orally showsthat the basic β-lactamase activity is low at the outset. Two to 4 hoursafter administration there was a clear increase in this activity,corresponding to the transit of the beads (FIG. 4).

The photos taken by scanning electron microscopy show the integrity ofthe bead at different places of the digestive tract.

The structure is slightly fragile in the small intestine and the insidewas completely destroyed at colonic level where the beads appearedcarriers of a cavity.

As illustrated in FIG. 5 the particles, having stayed in the stomach,looked very similar to those which had not undergone any treatment (FIG.2). In effect the surface had the same rugged and irregular look (FIGS.5A and 5B), owing to the presence of polyethylenimine, and thecross-section of the beads appeared uniform and dense (FIGS. 5C and 5D).

At the end of 2 h slight deformation of the beads became apparent (FIG.6A), but the particles still had the same surface appearance (FIG. 6B)and a dense cross-section (FIG. 6C), even though they were made a littlefragile by their stay in the small intestine (FIG. 6D).

On completion of transit, that is, 4 h after administration, the beadswere in the colon; the external appearance of the particles wasunchanged (FIG. 7A) with the same surface irregularities due to thepolyethylenimine (FIG. 7B). Yet the cross-section of the beads washollow (FIGS. 7C and 7D), due to the fact of degradation of the centralnetwork of calcium pectinate by the colonic pectinolytic enzymes.Finally, only the external shell formed by the polyethylenimineremained.

EXAMPLE 6 Encapsulation of Erythromycin Esterase

6.1 Production of a Soluble Fraction Containing Erythromycin Esterase

6.1.1. Operational Method

The culture was made from the strain of E. coli C600 pIP1100 from thePasteur Institute. The culture conditions were the following:inoculation of the Mueller-Hinton medium at 0.5% from a preculture ofabout 20 h, culture volumes of 200 or 400 mL in Erlenmeyer, fixedagitation at 150 rpm, temperature of 37° C.

A GOTS test helped establish that the strain produced much erythromycinesterase.

3.6 L of culture of E. coli C600 pIP1100 were concentrated according tothe following protocol:

Centrifuging for 15 min at 3400 g

Recovery of cap in potassium phosphate buffer 5 mM, pH 7.5, final volume70 mL

Second centrifuging of supernatant for 15 min at 3400 g

Recovery of cap in 20 mL of potassium phosphate buffer 5 mM, pH 7.5

Reuniting of caps of the 2 centrifuges (around 100 mL)

Washing of caps and centrifuging (10 min at 12,000 g)

Second centrifuging of supernatant (10 min 12,000 g)

Final volume of caps recovered in the potassium phosphate buffer: 100mL.

The erythromycin esterase was an intracellular enzyme. This is why itssolubilization required the cells to be broken. This operation wascarried out by ultrasonic extraction of centrifuging caps recovered inthe potassium phosphate buffer 5 mM, pH 7.5 according to the protocoldescribed hereinbelow.

Addition of 1% TritonX100 (v/v)

Cooling to 5° C.

Phonolysis 7 cycles of 1 min, initial temperature 5° C., maximaltemperature 15° C., power: 100% (500 W, 20 kHz) ; temperature taken to5° C. after each cycle

Centrifuging for 10 min at 12,000 g

Recovery of cap in 10 mL of potassium phosphate buffer 5 mM, pH 7.5

Recovery and congealing of the supernatant (91 mL)=solution A.

The erythromycin esterase activity was evaluated by the microbiologicaldosage in the supernatant and in the insoluble substances (cellulardebris) according to techniques known to the expert.

6.1.2. Results

The results are presented in Table 2. TABLE 2 Diameter of inhibition(mm) Sample T0 T30 T60 T120 Supernatant 31 25 18 — after ultrasound 21 —18 14 Cap after 30 28 24 — ultrasound 22.5 — 19.5 19

The erythromycin esterase activity was evaluated from the diameter ofinhibition.

The latter was 2 U/mL for the phonolysis supernatant and 1.5 U/mL forthe phonolysis cap (1 Unit (U)=1 μg of erythromycin degraded per min).

The balance of recuperation of the erythromycin esterase activity ispresented in Table 3 hereinbelow. TABLE 3 Estimated activity Totalestimated Sample (U/mL) Volume (mL) activity (U) Supernatant after 2.092 184 ultrasound Cap after ultrasound 1.5 10 15

The results clearly show that the essential element in the erythromycinesterase activity present has been solubilized in the phonolysis medium.

6.2 Encapsulation of Erythromycin Esterase

6.2.1. Operational Method

Encapsulation was achieved from the non-purified soluble fractionobtained after breaking the cells (solution A) according to thefollowing protocol.

Solubilization of 0.5 g of pectin in 10 mL solution A to obtain a finalconcentration of pectin of 5% (solution B). The pectin was added veryprogressively with magnetic stirring so as not to cause too many abruptvariations in pH. The pH was maintained in the region of 7 by additionof a few drops of soda 1M.

Dispersion of the solution of pectin (solution B) dropwise by means of aperistaltic pump to 40 mL of CaCl₂ at 6%. The beads thus formed werekept in the CaCl₂ for 20 min, recovered by Büchner filtration thenrinsed in demineralised water.

Reticulation of the beads by bath in a solution of PEI at 0.6% for 20min with magnetic stirring.

Recovery of the reticulated beads by filtration.

The beads were dried at ambient temperature (20° C.). 567 beads wereprepared in total with 6.1 mL of pectin/solution A mixture, for anactivity of 12.2 U.

The dried beads were disaggregated in a buffer HEPES/NaCl/EDTA 1%.

6.2.2. Results

The erythromycin esterase activity present in the initial solution ofpectin and that released in the disaggregation medium were dosedaccording to the same protocol as previously.

The results of the microbiological dosage are presented in Tables 4 and5. TABLE 4 Sample Average inhibition diameter (mm) Pectin/Solution A 23(solution B) - T0 Pectin/Solution A - T3h 19 Disaggregated beads - T0 24Disaggregated beads - T3h 18

TABLE 5 Sample Estimated activity Pectin (Solution B) 2.4 Disaggregatedbeads 2.2

The results show that the activity measured in the presence of pectin(solution B) is 2.4 U, while the theoretical activity present should bearound 12 U (6.1 mL at 2 U/mL, according to the dosage of erythromycinesterase in the phonolysis supernatant (Table 3).

The dosage of enzymatic activity of beads after disaggregation had beenestimated at 2.2 U; it representsed 90% of the initial activityintroduced to the beads.

These results help confirm unambiguously the presence of erythromycinesterase activity in the final fraction after encapsulation of theenzyme and disaggregation of the beads.

EXAMPLE 7 Encapsulation of DNA in the Calcium Pectinate Beads

7.1 Preparation of DNA

The active ingredient encapsulated here was a plasmid radiomarked withPhosphore 33. The radiomarking was done by means of the Nick TranslationKit N5500 from Amersham Biosciences according to the protocol describedby the supplier.

7.2 Encapsulation

7.2.1. Operational Method

The encapsulated DNA was either in free form, or complexed with cationiclipids (Lipoplexe) or a cationic polymer (Polyplexe) according to theoperational method described in Example 1.

For free DNA, around 5 μg of DNA radiomarked in solution in 750 μL ofMilliQ water were introduced to 0.75 g of a pectin solution, amidated ornot, at 10% so as to obtain a final concentration of pectin of 5%. Inthe case of the lipoplexes, 375 μL of an aqueous solution of radiomarkedDNA were mixed with 375 μL of a suspension of cationic liposomes (N/Pratio of 10). The 750 μL of resulting lipoplexes were then mixed with0.75 g of solution of pectin at 10% so as to obtain a finalconcentration of pectin of 5%.

In the case of polyplexes, 375 μL of an aqueous solution of radiomarkedDNA was mixed with 375 μL of an aqueous solution of PEI 4 mM. 375 μL ofthe suspension of polyplexes thus obtained were then mixed with 0.75 gof pectin solution at 10% to provide a final concentration of pectin of5%.

The beads of calcium pectinate encapsulating the free or complex DNAwere then prepared from solutions obtained hereinabove according to themethod described in Example 1.

The concentration of calcium chloride utilized here was 5% and that ofPEI for reticulation was 0.6%.

7.2.2. Results

They are illustrated in FIG. 8 which shows the encapsulation yields of aplasmid DNA in amidated or non-amidated pectin beads.

The encapsulated DNA was either in free form, or complexed in cationiclipids (Lipoplexe) or a cationic polymer (Polyplexe).

The encapsulation yields of DNA varied between 60 and 90% according tothe type of pectin used. They were generally more significant withamidated pectin. Complexing with lipids or a cationic polymer did notcause significant modifications to these yields, which remainedrelatively high.

1-11. (canceled)
 12. A drug delivery device for oral administration, andcolonic release, of an active agent, comprising: a) an active agentcapable of inactivating an antibiotic, and b) a drug delivery devicesuitable for administering the active agent to the colon.
 13. The drugdelivery device of claim 12, wherein the active agent is an enzymecapable of inactivating macrolide or quinolone antibiotics.
 14. The drugdelivery device of claim 13, wherein the enzyme capable of inactivatingmacrolide antibiotics is erythromycin esterase.
 15. The drug deliverydevice of claim 12, wherein the device comprises beads of pectin in theform of a cationic salt enclosing the active agent.
 16. The drugdelivery device of claim 15, wherein the pectin is reticulated by acationic polymer.
 17. The drug delivery device of claim 15, wherein thepectin salt is a calcium pectinate.
 18. The drug delivery device ofclaim 15, wherein the pectin is an amidated pectin.
 19. A method ofreducing the concentration of an antibiotic in the colon of a patient,comprising orally administering the drug delivery device of claim 1 to apatient who has been, is being, or will be administered an antibiotic.20. The method of claim 19, wherein the active agent in the drugdelivery device is an enzyme capable of inactivating macrolide orquinolone antibiotics.
 21. The method of claim 20, wherein the enzymecapable of inactivating macrolide antibiotics is erythromycin esterase.22. The method of claim 19, wherein the device comprises beads of pectinin the form of a cationic salt enclosing the active agent.
 23. Themethod of claim 22, wherein the pectin is reticulated by a cationicpolymer.
 24. The method of claim 22, wherein the pectin salt is acalcium pectinate.
 25. The method of claim 22, wherein the pectin is anamidated pectin.
 26. A method of preparing a drug delivery device fororal administration, and colonic delivery, of an active agent thatinactivates an antibiotic, comprising: a) preparing a 4-10% (m/v) pectinsolution that includes an active agent that inactivates an antibiotic,b) adding the pectin solution to a 2-10% (m/v) calcium chloride solutionto form pectin cationically crosslinked beads, and c) reticulating thepectin beads with a 0.5-2% (m/v) polyethylenimine solution.
 27. Themethod of claim 26, wherein the pectin solution further comprises asecond active agent, where the second active agent is an antibiotic, ananti-inflammatory compound, an anti-histamine, an anti-cholinergic, anantiviral, an antimitotic, a peptide, a protein, a gene, an anti-senseoligonucleotide, a diagnostic agent, an immunosuppressive agent or abacteria.
 28. A drug delivery device comprising an active agent capableof inactivating a macrolide, tetracycline or quinolone antibiotic. 29.The drug delivery device of claim 28, wherein the device is suitable foradministering the active agent to the colon.
 30. The drug deliverydevice of claim 28, wherein the active agent is an enzyme capable ofinactivating macrolide or quinolone antibiotics.
 31. The drug deliverydevice of claim 30, wherein the enzyme capable of inactivating macrolideantibiotics is erythromycin esterase.
 32. The drug delivery device ofclaim 28, wherein the device comprises beads of pectin in the form of acationic salt enclosing the active agent.
 33. The drug delivery deviceof claim 32, wherein the pectin is reticulated by a cationic polymer.34. The drug delivery device of claim 32, wherein the pectin salt is acalcium pectinate.
 35. The drug delivery device of claim 32, wherein thepectin is an amidated pectin.
 36. The drug delivery device of claim 28,further comprising a second active agent, wherein the second agent is anantibiotic, an anti-inflammatory compound, an anti-histamine, ananti-cholinergic, an antiviral, an antimitotic, a peptide, a protein, agene, an anti-sense oligonucleotide, a diagnostic agent, animmunosuppressive agent or a bacteria.
 37. A method of reducing theconcentration of a macrolide, tetracycline or quinolone antibiotic inthe colon of a patient, comprising orally administering an effective,antibiotic-reducing amount of the drug delivery device of claim 28 to apatient who has been, is being, or will be administered a macrolide,tetracycline or quinolone antibiotic.
 38. The method of claim 37,wherein the drug delivery device administers the active agent to thecolon.
 39. The method of claim 37, wherein the active agent in the drugdelivery device is an enzyme capable of inactivating macrolide orquinolone antibiotics.
 40. The method of claim 39, wherein the enzymecapable of inactivating macrolide antibiotics is erythromycin esterase.41. The method of claim 37 wherein the device comprises beads of pectinin the form of a cationic salt enclosing the active agent.
 42. Themethod of claim 41, wherein the pectin is reticulated by a cationicpolymer.
 43. The method of claim 41, wherein the pectin salt is acalcium pectinate.
 44. The method of claim 41, wherein the pectin is anamidated pectin.
 45. A drug delivery device comprising: a) a pectin andb) an active agent capable of inactivating an antibiotic.
 46. The drugdelivery device of claim 45, wherein the device is suitable foradministering the active agent to the colon.
 47. The drug deliverydevice of claim 45, wherein the active agent is an enzyme capable ofinactivating macrolide or quinolone antibiotics.
 48. The drug deliverydevice of claim 47, wherein the enzyme capable of inactivating macrolideantibiotics is erythromycin esterase.
 49. The drug delivery device ofclaim 45, further comprising a metal cation.
 50. The drug deliverydevice of claim 49, wherein the cation is a calcium ion.
 51. The drugdelivery device of claim 50, further comprising a cationic polymer. 52.The drug delivery device of claim 45, wherein the device comprises beadsof pectin in the form of a cationic salt enclosing the active agent. 53.The drug delivery device of claim 52, wherein the pectin is reticulatedby a cationic polymer.
 54. The drug delivery device of claim 52, whereinthe pectin salt is a calcium pectinate.
 55. The drug delivery device ofclaim 52, wherein the pectin is an amidated pectin.
 56. A method ofreducing the concentration of an antibiotic in the colon of a patient,comprising orally administering an effective, antibiotic-reducing amountof the drug delivery device of claim 45 to a patient who has been, isbeing, or will be administered an antibiotic.
 57. The method of claim56, wherein the drug delivery device comprises an enzyme capable ofinactivating macrolide or quinolone antibiotics.
 58. The method of claim57, wherein the enzyme capable of inactivating macrolide antibiotics iserythromycin esterase.
 59. A drug delivery device comprising: a) a firstactive agent capable of inactivating an antibiotic, and b) a secondactive agent, where the second active agent is an antibiotic, ananti-inflammatory compound, an anti-histamine, an anti-cholinergic, anantiviral, an antimitotic, a peptide, a protein, a gene, an anti-senseoligonucleotide, a diagnostic agent, an immunosuppressive agent or abacteria.
 60. The drug delivery device of claim 59, wherein the deviceis suitable for administering the active agents to the colon.
 61. Thedrug delivery device of claim 59, wherein the first active agent is anenzyme capable of inactivating macrolides or quinolones.
 62. The drugdelivery device of claim 61, wherein the enzyme capable of inactivatingmacrolides is erythromycin esterase.
 63. The drug delivery device ofclaim 59, wherein the device comprises beads of pectin in the form of acationic salt enclosing the active agents.
 64. The drug delivery deviceof claim 63, wherein the pectin is reticulated by a cationic polymer.65. The drug delivery device of claim 59, wherein the second activeagent is specific for treating ulcerative colitis or Crohn's disease.66. A method of reducing the concentration of an antibiotic in the colonof a patient, comprising orally administering an effective,antibiotic-reducing amount of the drug delivery device of claim 60 to apatient who has been, is being, or will be administered an antibiotic.67. The method of claim 66, wherein the drug delivery device comprisesan enzyme capable of inactivating macrolides or quinolones.
 68. Themethod of claim 67, wherein the enzyme capable of inactivatingmacrolides is erythromycin esterase.
 69. The method of claim 66, whereinthe second active agent is specific for treating ulcerative colitis orCrohn's disease.